This invention relates to a method of making an alcoholic beverage by fermentation, and to a fermentation test kit for use in such method.

During the fermentation of, for example, 5 grape musts, the wine yeasts responsible for fermentation require the presence of sugar and free amino nitrogens (also referred to as "FAN") as essential nutrients for fermentation. When making wine from musts that have a sub-optimal FAN content, lagging

10 (i.e. slow, retarded, or stuck) fermentations accompanied by the formation of off-flavours are often encountered, which result in poor wine quality. It is known for deficiencies in FAN levels to be rectified by the addition of diammonium phosphate (also referred to

15. as "DAP") . DAP is a source of assimilable nitrogen and, when added in sufficient quantities, effectively restores any deficiency in the nutrient requirement of yeasts during fermentation.

Traditional techniques (such as the ninhydrin

20 methodology) of controlling the FAN levels in grape musts involve a determination of assimilable nitrogen levels in the musts, and adjustments to make up for any deficiencies, prior to fermentation. The ninhydrin determination of assimilable nitrogen levels requires

25 specialized knowledge and the use of expensive apparatus in controlled laboratory conditions, which are normally well beyond the ability and means of the average wine maker.

It is an object of the present invention to

30 provide a method of making an alcoholic beverage, and more particularly wine, which enables the FAN content of the fermenting liquor to be adjusted to appropriate levels in a manner that is inexpensive and can readily be implemented by the average maker of such beverage,

35 without the need for specialized knowledge or expensive

apparatus and access to laboratory facilities.

According to the invention there is provided a method of making an alcoholic beverage by fermentation of a fermentable liquor, in which a source of assimilable nitrogen is added to the liquor to supplement the assimilable nitrogen content of the liquor, wherein gas evolving from the liquor during fermenta ion is monitored for the presence therein of H ₂ S, and wherein said source of assimilable nitrogen is added to the liquor in response to the detection of H,S in said gas .

The method may comprise the steps of (a) monitoring said gas for the presence therein of H,S, for a predetermined period, (b) thereupon adding a portion of said source of assimilable nitrogen to the liquor in response to the detection of H _: S in said gas -during that period, and (c) repeating the steps (a) and (b) .

The steps (a) and (b) may be repeated continuously, from the beginning to the end of the fermentation.

Said gas may be monitored continuously during each said period.

The steps (a) and (b) may be repeated at least once a day.

Where the fermentation is a relatively cold or slow fermentation, the steps (a) and (b) may be repeated once a day. Where the fermentation is a relatively warm or vigorous fermentation, the steps (a) and (b) may be repeated at least once every 8 hours. Said gas may be monitored for the presence therein of H,S by exposing an indicator strip which is selectively responsive to the presence of H _: S, to the gas . The indicator strip may be a zinc acetate impregnated indicator strip, and may, after exposure to said gas, be reacted with a first reagent comprising a

solution of 4-amino-N, N-dimethylaniline sulphate (or its hydrochloride) , to which a second reagent comprising a solution of ferric chloride is added, whereby the development of a blue colour in the admixture of said reagents establishes the presence of H,S in said gas.

The invention extends to a fermentation test kit for use in making an alcoholic beverage by fermentation of a fermentable liquor, which includes means for detecting the presence of H,S in gas evolving from the liquor during fermentation. Said means may be in the form of a supply of indicator strips which are selectively responsive to the presence of H ₂ S.

The indicator strips may be zinc acetate impregnated indicator strips, the kit further comprising two reagents, namely a first reagent _^ comprising a solution of 4-amino-N, N-dimethylaniline sulphate (or its hydrochloride) , and a second reagent comprising a solution of ferric chloride. The invention will now be described in more detail, by way of example, with reference to the accompanying drawings .

In the drawings : Figure 1 illustrates the mechanism of H,S development during fermentation;

Figure 2 illustrates a method of making wine in accordance with the invention,- and

Figure 3 illustrates a fermentation test kit in accordance with the invention. As illustrated in Figure 1, yeast metabolism requires, as essential nutrients in the fermenting liquor, assimilable sugars and assimilable nitrogens or FAN (amino acids, lower peptides, and ammoniacal nitrogen) . Under conditions of sub-optimal FAN content, the proteolytic activity of the yeast is stimulated. This causes the yeast to act on sources of non-

assimilable nitrogen (proteins and higher peptides in musts and other fermenting liquors) , to form the required assimilable nitrogen through proteolytic degradation. Through this activity, the sulphur derivatives of protein (e.g. cysteine) are desulphurised and liberated as H,S . It follows that there is an inverse relationship between the assimilable nitrogen content of the fermenting liquor and the development of K,S during fermentation of the liquor. The presence of H,S in the gas evolving from the fermenting liquor is a direct result of assimilable nitrogen deficiency experienced by the yeast, and is thus directly related to the nitrogen demand of the particular yeast when the assimilable nitrogen content of the liquor is at a sub-optimal level.

Referring now to Figure 2, reference numeral 10 indicates a fermentation vessel in which grape musts 12 are fermented. The vessel has a manhole 14, and a duct 16 via which gases evolving from the fermenting liquor can leave the vessel.

In implementing the present invention the manhole 14 is opened periodically, an unexposed indicator strip 18 suspended in the space above the fermenting liquor 12, and the manhole closed again. The indicator strip is thus continuously exposed to the stream of gas evolving from the fermenting liquor. Each time a new, unexposed indicator strip is inserted in the vessel, the previously inserted, now exposed, indicator strip is removed from the vessel. Each indicator strip should preferably be exposed to the gas evolving from the fermenting liquor for a continuous period of up to 24 hours. In the case of slower or colder fermentations exposure for a continuous period of 24 hours, and replacement of the strip once a day, is recommended. In the case of more vigorous or warmer fermentations exposure for a continuous period of up to 8 hours, and replacement of the strip at leaεt three

times a day, is recommended.

The indicator strips 18 are plastic strips which have a piece of filter paper at their tips, the strips having been sensitised by impregnation of the filter paper with a 6% solution of zinc acetate (Zn(CH ₃ COO) ₂ .2H,0) and subsequent drying.

When inserting a new, unexposed indicator strip in the vessel the previously, now exposed, indicator strip is removed from the vessel and developed. This is done by inserting the indicator strip into a test tube 20 which contains 5m of a first reagent, to which 5 drops of a second reagent are added by means of a dropper 22. The first reagent comprises a 0,02% solution of 4-amino-N, -dimethylaniline sulphate (or its hydrochloride) in 0, IN HCl, and the second reagent comprises a 2,7% solution of ferric chloride (FeCl ₃ .6H ₂ 0) in 0, IN HCl. The contents of the test tube are shaken vigorously and then left for 30 minutes for maximum colour development. Development of a blue colour (methylene blue) is a positive indication of the presence of sulphide and thus a nitrogen deficiency of the particular fermenting liquor. The colour intensity is indicative of the degree of nitrogen deficiency (and hence the nitrogen demand of the particular yeast strain) . If the test indicates a nitrogen deficiency, a predetermined quantity of DAP or other suitable source of assimilable nitrogen is added to the liquor 12, as indicated at 24. The amount of DAP added may vary according to the colour intensity obtained when the indicator strip 18 is developed. For example, a colour chart may be provided to enable the wine maker to differentiate between light blue, intermediate blue, and dark blue, and lOg/h , 20g/h , or 30g/h^ of DAP added depending on whether light, intermediate, or dark blue is indicated. Normally, no more than 30g/h^ of DAP should be added at a time, as this may result in over-dosing.

The more frequent the above procedure is repeated, the less will the FAN content of the fermenting liquor deviate from the optimal value. It has been found that, with colder, slower fermentation processes such as the ones required for making white wine, a frequency of once per day (exposure of the strips for a continuous period of 24 hours) gives good results. With warmer, faster fermentation processes such as the ones required for making red wine, however, a frequency of 3 times per day (exposure of the strips for a continuous period of 8 hours) may be required

Table 1 at the end of the specification indicates the results of tests carried out on six of the most popular yeast strains used in South Africa From this it is evident that, according to the traditional method, the nitrogen-deficient control samples required the addition of 45g/hf DAP prior to fermentation When compared to control samples to which no DAP was added, the addition of DAP according to the traditional method resulted in an average decrease of 58,8% in the formation of hydrogen sulphide (i.e the nitrogen demand of the yeast strains) In contrast, the new method required an average addition of only 33g/h DAP and resulted m an average decrease of 81,7% m the formation of hydrogen sulphide.

It is evident from column 2 m Table 1 that there are significant differences m tne production of hydrogen sulphide among individual strains (i.e. differences in their nitrogen demand) In the case of traditional methods these differences are not taken into consideration In the new method, however, the nitrogen demand of each particular yeast strain s catered for as fermentation progresses The new method therefore recognises the most important variables that influence the metabolic functions of a particular fermentation.

The materials for enabling the wine maker to

implement the method may be provided in the form of a fermentation test kit, such as the one illustrated at 30 in Figure 3. The kit 30 comprises a tube 32 containing a supply of sensitised indicator strips 18, the tube being closed in an air-tight manner by means of a cap which includes a desiccator 34, one or more containers 36 containing a supply of the first reagent, and a container 38 for containing a supply of the second reagent. The kit 30 may further include a test tube and a dropper such as the ones illustrated in Figure 2.

By implementing the technique as outlined above in the making of wine, the wine maker can detect sub-optimal conditions as soon as they occur, immediately take remedial action, and within a short period thereafter detect the effect of the remedial action by exposing a new indicator strip to the gas evolving from the fermenting liquor for the recommended period and repeating the test. He can moreover do so in a relatively simple and inexpensive manner, without the need for specialized knowledge or access to expensive laboratory facilities, and without having any knowledge of the actual FAN content of the musts . Thus, the technique enables him, in response to detecting the presence of H ₂ S, to add small quantities, of DAP at a time, to monitor the effect of such additions, and to continue the procedure until just enough DAP has been added for signs of the presence of H,S to disappear. As a consequence, over-dosing with DAP is avoided, and there is no need to determine the FAN content prior to fermentation.

A large number of genetically different yeast strains for fermentation of grape musts are commercially available. The demand for assimilable nitrogen of different strains varies significantly. The method of the present invention recognises and accommodates the individual demand of each particular

yeast strain. This is a very important additional advantage which cannot be achieved when adjustments for nitrogen deficient musts are made prior to fermentation. A further advantage of the technique is that the cooling requirements of the fermenting musts are significantly reduced. In existing techniques the quantity of DAP that has to be added for optimal fermentation (as determined by laboratory analysis) is normally all added at the beginning of the fermentation process. The abundance, as a result, of FAN in the fermenting musts at the early stages of fermentation leads to a highly increased fermentation activity which, in turn, leads to excessive generation of heat. This heat has to be removed from the fermenting musts by cooling. With the technique of the present invention there is only a moderate increase in fermentation activity, thus limiting the generation of heat . The method of the present invention applies to all fermented alcoholic beverages.

SUBSTITUTE SHEET (RUL£ 26)

TABLE 1

H ₂ S FORMATION AT 1 5 °C RELATIVE ABSORPTION UNITS

1 2 3 3A 4 4A

TRADITIONAL NEW METHOD

STRAIN CONTROL FAN DAP FAN DAP

N 96 4,099 1 , 167 45 1 ,91 5 20

VIN 1 3 23, 160 2,627 45 2,373 1 5

VIN 7 44, 141 1 2,031 45 4,229 5

WE 372 49,260 19,069 45 13,260 35

WE 14 103,425 75,641 45 38,654 60

228 203,764 78, 197 45 22,740 65

AVERAGE 75,808 31 ,455 45 13,861 33

%REDUCTION -58,5 -81 ,7 -26

Column 1 : Yeast strain

Column 2: Control (H ₂ S values).

Column 3: Traditional method of determining the nitrogen demand of yeast strains prior to fermentation (H ₂ S values). Column 3A: Diammonium phosphate additions in respect of traditional method in g/hf . Column 4: New method of determining the nitrogen demand of yeast strains during fermentation (h ₂ S values). Column 4A: Diammonium phosphate additions in respect of new method in g/h ? .